A caspase-8-independent component in TRAIL/Apo-2L-induced cell death in human rhabdomyosarcoma cells.

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) belongs to the Tumor necrosis factor (TNF) family of death-inducing ligands, and signaling downstream of TRAIL ligation to its receptor(s) remains to be fully elucidated. Components of the death-inducing signaling complex (DISC) and TRAIL signaling downstream of receptor activation were examined in TRAIL - sensitive and -resistant models of human rhabdomyosarcoma (RMS). TRAIL ligation induced DISC formation in TRAIL-sensitive (RD, Rh18, Rh30) and TRAIL-resistant RMS (Rh28, Rh36, Rh41), with recruitment of FADD and procaspase-8. In RD cells, overexpression of dominant-negative FADD (DNFADD) completely abolished TRAIL-induced cell death in contrast to dominant-negative caspase- 8 (DNC8), which only partially inhibited TRAIL-induced apoptosis, growth inhibition, or loss in clonogenic survival. DNC8 did not inhibit the cleavage of Bid or the activation of Bax. Overexpression of Bcl-2 or Bcl-xL inhibited TRAIL-induced apoptosis, growth inhibition, and loss in clonogenic survival. Bcl-2 and Bcl-xL, but not DNC8, inhibited TRAIL-induced Bax activation. Bcl-xL did not inhibit the early activation of caspase-8 (<4 h) but inhibited cleavage of Bid, suggesting that Bid is cleaved downstream of the mitochondria, independent of caspase-8. Exogenous addition of sphingosine also induced activation of Bax via a caspase-8-and Bid-independent mechanism. Further, inhibition of sphingosine kinase completely protected cells from TRAIL-induced apoptosis. Data demonstrate that in RMS cells, the TRAIL signaling pathway circumvents caspase-8 activation of Bid upstream of the mitochondria and that TRAIL acts at the level of the mitochondria via a mechanism that may involve components of the sphingomyelin cycle.

Hypermethylation of the gene promoter and enhancer region can regulate Fas expression and sensitivity in colon carcinoma.

Expression of the cell surface receptor Fas is frequently lost or decreased during tumor progression in human colon carcinomas. The methylation status of a 583 bp CpG-rich region within the Fas promoter (-575 to +8) containing 28 CpG sites was determined in human colon carcinoma cell lines. In Caco(2) (no Fas expression), 82-93% of CpG sites were methylated, whereas none were methylated in GC(3)/c1 (high Fas expression). In RKO (intermediate level of Fas), a single CpG site, located at -548, was 100% methylated. The inhibitor of methylation, 5-aza-2'-deoxycytidine (5-azadC), upregulated Fas expression in four of eight cell lines, and sensitized RKO cells to recombinant FasL-induced apoptosis. The p53-binding region in the first intron of the Fas gene was partially methylated in Caco(2), and 5-azadC potentiated Ad-wtp53-induced upregulation of Fas expression. Methylation-specific PCR of the first intron detected partial methylation in four out of 10 colon carcinoma tumor samples in vivo. The data suggest that DNA hypermethylation is one mechanism that contributes to the downregulation of Fas expression and subsequent loss of sensitivity to Fas-induced apoptosis in colon carcinoma cells.

Safety and efficacy of combined high-dose treatment with calcipotriol ointment and solution in patients with psoriasis.

BACKGROUND: In the vast majority of psoriatic patients, psoriatic lesions are localised on the body as well as on the scalp. Therefore, safety data on the combined use of calcipotriol in lotion and calcipotriol in ointment are needed. OBJECTIVE: This study investigated the effect of high-dose treatment with a combination of calcipotriol ointment and scalp solution on calcium metabolism, indices of bone turnover and PASI in patients with extensive psoriasis. METHODS: Following a 2-week wash-out period, 88 patients were randomised to 4 weeks of treatment with either calcipotriol ointment/scalp solution (80-100 g/week and 30-50 ml/week, respectively; n = 41) or with a dithranol/tar regimen (n = 47). Patients were seen at weeks 1, 2 and 4 during treatment and 1 week following cessation of treatment. RESULTS: No significant differences at the end of treatment were found between the 2 groups with respect to 24-hour urinary excretion of calcium (expressed as calcium/creatinine ratio), phosphate or pyridinoline, serum concentrations of calcium (albumin corrected), creatinine, phosphate, parathyroid hormone, 25-hydroxyvitamin D(3), 1,25-dihydroxyvitamin D(3), osteocalcin, alkaline phosphatase (total and bone-specific iso-enzymes) or 1-collagen telopeptide. At the end of treatment, the psoriasis area and severity index had decreased by 57.4% in the calcipotriol group and by 36.1% in the dithranol/tar group (p = 0.004). Investigators' and patients' assessments of overall efficacy also favoured treatment with calcipotriol (p < 0.001). CONCLUSION: The combined use of calcipotriol ointment/scalp solution did not affect the indices of calcium metabolism or bone turnover and was significantly more effective than dithranol/tar in reducing disease severity and extent in patients with extensive psoriasis.

Identification of a novel psoriasis susceptibility locus at 1p and evidence of epistasis between PSORS1 and candidate loci.

The pathogenesis of all forms of psoriasis remains obscure. Segregation analysis and twin studies together with ethnic differences in disease frequency all point to an underlying genetic susceptibility to psoriasis, which is both complex and likely to reflect the action of a number of genes. We performed a genome wide analysis using a total of 271 polymorphic autosomal markers on 284 sib relative pairs identified within 158 independent families. We detected evidence for linkage at 6p21 (PSORS1) with a non-parametric linkage score (NPL)=4.7, p=2 x 10(-6) and at chromosome 1p (NPL=3.6, p=1.9 x 10(-4)) in all families studied. Significant excess (p=0. 004) paternal allele sharing was detected for markers spanning the PSORS1 locus. A further three regions reached NPL scores of 2 or greater, including a region at chromosome 7 (NPL 2.1), for which linkage for a number of autoimmune disorders has been reported. Partitioning of the data set according to allele sharing at 6p21 (PSORS1) favoured linkage to chromosomes 2p (NPL 2.09) and 14q (NPL 2.0), both regions implicated in previous independent genome scans, and suggests evidence for epistasis between PSORS1 and genes at other genomic locations. This study has provided linkage evidence in favour of a novel susceptibility locus for psoriasis and provides evidence of the complex mechanisms underlying the genetic predisposition to this common skin disease.

p53 dependence of Fas induction and acute apoptosis in response to 5-fluorouracil-leucovorin in human colon carcinoma cell lines.

We examined the patterns of induction of apoptosis, Fas expression, and the influence of the status of the p53 tumor suppressor gene, in response to treatment of human colon carcinoma cell lines to 5-fluorouracil (FUra) combined with leucovorin (LV) under conditions of both DNA-directed (HT29, VRC5/c1, and RKO) and RNA-directed (HCT8 and HCT116) cytotoxicity. Acute apoptosis was induced in cell lines expressing wtp53 (RKO, HCT8, and HCT116), independent of the mechanism of FUra action. In HT29 cells that expressed mp53, apoptosis was a delayed event. Cell lines undergoing DNA-directed FUra cytotoxicity demonstrated marked accumulation of cells in S-phase (HT29 and RKO), whereas those lines undergoing RNA-directed cytotoxicity (HCT8 and HCT116) demonstrated marked cell cycle phase arrest in G2-M, both reversible by dThd. dThd partially protected HCT8 and HCT116 cells from FUra-LV-induced apoptosis but had no influence on FUra-LV-induced loss in clonogenic survival. In cells expressing wtp53, the Fas death receptor was induced in response to FUra-LV treatment. FUra-LV sensitized RKO cells to the anti-Fas monoclonal antibody CH-11 that was completely reversed by dThd, demonstrating the involvement of DNA damage in FUra-LV-induced, Fas-dependent sensitization to CH-11. In contrast, FUra-LV sensitized HCT116 cells to CH-11-induced apoptosis, which was not dThd reversible. Transduction of HT29 cells with Ad-wtp53 induced elevated Fas expression and sensitized the cells to FUra-LV-induced apoptosis. Data indicate that the presence of a wtp53 gene determines FUra-LV-induced Fas expression, the kinetics of FUra-LV-induced apoptosis and not the extent of apoptosis induced, both being independent of the mechanism of FUra action. Therefore, in colon carcinomas that express wtp53, the approach to sensitize tumors to Fas-mediated apoptosis may be further enhanced from the effect of FUra-LV in elevating Fas expression in a p53-dependent manner.

Effectiveness and economic impact associated with a program for outpatient management of acute deep vein thrombosis in a group model health maintenance organization.

BACKGROUND: Controlled clinical trials have demonstrated that outpatient administration of low-molecular-weight heparin to patients with acute deep vein thrombosis (DVT) provides safety and efficacy equivalent to that of traditional inpatient therapy with unfractionated heparin. Whether favorable results reported in controlled clinical trials are achievable in clinical practice is an important consideration. METHODS: Appropriate patients with objectively diagnosed DVT were treated as outpatients with low-molecular-weight heparin and warfarin sodium according to an approved guideline. The primary end point for analysis consisted of objectively diagnosed symptomatic recurrent thromboembolism or major bleeding within a 90-day evaluation period. The incremental cost incurred by the organization while using the outpatient DVT treatment guideline was determined. Incremental cost savings of the outpatient DVT treatment program were determined based on the cost that would have accrued had the patient been admitted to the hospital for treatment with unfractionated heparin. RESULTS: We enrolled 391 patients (91.4%) in the outpatient DVT treatment program. Of these, 373 (95.4%) completed 90 days of therapy without reaching the primary end point. The percentage of patients reaching the primary outcome measure (4.6%) fell within the range of patients enrolled in controlled clinical trials (3.5%-9.4%). During the 2-year program evaluation, total cost savings of $1,108,587 were realized. CONCLUSIONS: Outpatient treatment of acute DVT can be managed safely and effectively in clinical practice. The potential savings associated with outpatient DVT treatment are substantial. Arch Intern Med. 2000;160:2926-2932

Pediatric rhabdomyosarcoma cell lines are resistant to Fas-induced apoptosis and highly sensitive to TRAIL-induced apoptosis.

Seven pediatric rhabdomyosarcoma (RMS) cell lines were resistant to the induction of apoptosis via the Fas death receptor. In contrast, four of seven lines (RD, Rh1, Rh18, and Rh30) were highly sensitive to tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). TRAIL induced apoptosis within 4 h and also reduced clonogenic survival, both reversible by caspase inhibitors. DR5 (but not DR4) was expressed at high level in all cell lines. Expression of the decoy receptors DcR1 and DcR2 did not correlate with TRAIL sensitivity. All RMS lines expressed the adapter molecule FADD, and six of seven expressed procaspase-8. Expression of the inhibitory proteins c-FLIPL and c-FLIPs was high in three TRAIL-sensitive (RD, Rh1, and Rh30) and two TRAIL-resistant (Rh28 and Rh41) lines. All RMS lines expressed Bid and procaspases-3, -6, -7, and -9. Procaspases-8 and -10 were highest in TRAIL-sensitive RMS (RD, Rh1, and Rh30), and procaspase-10 was not expressed in Rh18, Rh36, or Rh41. TRAIL induced loss of mitochondrial membrane potential in TRAIL-sensitive Rh1 but not in TRAIL-resistant Rh41 cells. There was no correlation between expression of members of the Bcl-2 family (Bcl-2, Bcl-xL, Bax, and Bak) and TRAIL sensitivity. TRAIL-sensitive Rh18 expressed procaspase-8 in the absence of procaspase-10 and c-FLIP, and procaspase-10 was not detected in TRAIL-resistant Rh41 in the presence of procaspase-8 and c-FLIP. Data suggest that caspase-8 may be sufficient to deliver the TRAIL-induced apoptotic signal in the absence of both caspase-10 and c-FLIP (Rh18) but not in the presence of c-FLIP (Rh41). In RD, Rh1, and Rh30, the presence of c-FLIP may require amplification of the apoptotic signal via caspase-10.

Randomized, placebo-controlled trial of oral phytonadione for excessive anticoagulation.

STUDY OBJECTIVE: To compare the efficacy of managing excessive anticoagulation in the absence of bleeding by either omitting warfarin therapy alone or administering oral phytonadione in addition to omitting warfarin therapy. DESIGN: Randomized, double-blind, placebo-controlled study. SETTING: Clinical pharmacy anticoagulation service in a group model health maintenance organization. SUBJECTS: Thirty nonbleeding patients with international normalized ratios (INRs) ranging from 6.0-10.0. INTERVENTIONS: Patients were randomized to receive either a single oral dose of phytonadione 2.5 mg or placebo. Both groups omitted warfarin doses until the INR became less than or equal to 4.0. MEASUREMENTS AND RESULTS: The mean calculated time to reach an INR of 4.0 was significantly greater in the placebo than the phytonadione group (2.6 vs 1.4 days, p=0.006). Overcorrection of anticoagulation was significantly more common in patients receiving phytonadione. Overt warfarin resistance was not observed in either group after reinitiating warfarin therapy. No major bleeding or thromboembolic complications occurred, and minor bleeding episodes were similar in both groups. CONCLUSION: The addition of oral phytonadione 2.5 mg reduced the time to achieve an INR of 4.0 by approximately 1 day compared with omitting warfarin therapy alone. Adverse events did not differ between the two groups. Both strategies were effective in managing asymptomatic patients with INRs of 6.0-10.0. Oral phytonadione may be most appropriate for patients at high risk for bleeding in whom the benefit of prompt INR reduction would outweigh the thromboembolic risk associated with INR overcorrection.

Fas-dependent and -independent mechanisms of cell death following DNA damage in human colon carcinoma cells.

In thymidylate synthase-deficient (TS-) colon carcinoma cells, thymineless death is mediated via Fas/Fas ligand (FasL) interactions after thymidine deprivation and inhibited by the Fas-inhibitory monoclonal antibody NOK-1. The objective of the study was to elucidate whether other modes of DNA damage induced by doxorubicin, topotecan, and etoposide (VP-16) could elicit a similar cytotoxic response in TS- cells by signaling via the Fas death receptor. After a 72-h drug exposure, a loss in clonogenic survival that was not prevented by NOK-1 was induced by each agent in the absence of acute apoptosis, yielding IC50 values of 5 (doxorubicin), 10 (topotecan), and 150 nM (VP-16). Furthermore, TS- cell clones selected for resistance to Fas-mediated apoptosis (CH-11) were cross-resistant to the induction of thymineless death after thymidine deprivation but were not cross-resistant to doxorubicin, topotecan, or VP-16. A close correlation was found between acute induction of apoptosis (24 h) and up-regulated expression of FasL at high concentrations of each of the three agents (0.3-3 microM doxorubicin, 0.3-3 microM topotecan, and 10-90 microM VP-16), which was caspase dependent but Fas independent. At all drug concentrations, cell cycle distribution analyses demonstrated marked accumulation of cells in the G2-M phase. At nanomolar drug concentrations, prolonged arrest of TS- cells in G2-M phase resulted in the up-regulation of FasL expression and the delayed appearance of apoptotic cells (6 days), which could also be inhibited by the general caspase inhibitor Z-VAD-FMK, but not by NOK-1 or Fas-Fc. In clonogenic assays, Z-VAD-FMK did not rescue cells treated with VP-16 in contrast to treatment with CH-11 or thymineless stress, suggesting an irreversible commitment to cell death in G2-M phase. Expression of FasL at all drug concentrations appeared to be unrelated to the mechanism of drug-induced apoptosis. This was in contrast to the Fas-dependent regulation of thymineless death, which could be inhibited by blocking Fas/FasL interactions.

The efficacy of narrowband ultraviolet B phototherapy in psoriasis using objective and subjective outcome measures.

The efficacy of narrowband ultraviolet B (UVB) was assessed in 100 consecutive patients with psoriasis by quantifying disease severity using objective (Psoriasis Area and Severity Index, PASI and Dermatologists Global Assessment, DGA) and subjective (Psoriasis Disability Index, PDI) measures. The median pretreatment PASI, DGA and PDI were 5.7 (interquartile range, IQR 4.5-8.35), 7 (IQR 6-9) and 42 (IQR 29-63.5), respectively. At 3 month follow-up, the PASI, DGA and PDI had fallen to 2.7 (IQR 1.1-3.5), 3 (IQR 2-5) and 30 (IQR 21-50.5), respectively (P < 0.001). A small group of patients continued to score highly on their PDI despite being clinically clear or having minimal disease, possibly representing chronic disability behaviour. Patients exhibiting this may require more intensive supervision. In most patients, symptoms of itch and pain improved or disappeared (70% and 75%, respectively). Side-effects were reported in 18%. Narrowband UVB phototherapy is safe and effective for psoriasis. Symptoms and subjective quality of life measures improved significantly. Both objective and subjective measures should be used when evaluating the efficacy of a treatment for psoriasis.

A Fas-dependent component in 5-fluorouracil/leucovorin-induced cytotoxicity in colon carcinoma cells.

We have shown previously (J. A. Houghton et al., Proc. Natl. Acad. Sci. USA, 94: 8144-8149, 1997) that thymineless death in thymidylate synthase-deficient (TS-) colon carcinoma cells is mediated via Fas/FasL interactions after deoxythymidine (dThd) deprivation, and that Fas-dependent sensitivity of human colon carcinoma cell lines may be dependent upon the level of Fas expressed. The objective of this study was to elucidate whether a Fas-dependent component exists in 5-fluorouracil (FUra)/leucovorin (LV)-induced cytotoxicity of colon carcinoma cells, and whether this may be potentiated by IFN-gamma-induced elevation in Fas expression, using the HT29 cell line as a model. The cytotoxic activity of FUra/LV was inhibited by dThd in HT29 cells and also, in part, by NOK-1+NOK-2 MoAbs that prevent Fas/FasL interactions. FUra/LV-induced cytotoxicity was significantly potentiated by IFN-gamma, reversed by exposure to NOK-1+NOK-2 antibodies, and correlated with a 4-fold induction of Fas expression in the presence of IFN-gamma and significant elevation in expression of FasL. Using five additional human colon carcinoma cell lines, FUra/LV-induced cytotoxicity was dThd-dependent in GC3/c1, VRC5/c1, and Caco2 but not in HCT8 or HCT116 cells. Like HT29 cells, this cytotoxicity was potentiated by IFN-gamma in GC3/c1 and VRC5/c1 but not in Caco2, which fails to express Fas, nor in HCT8 and HCT116, in which no dThd-dependent FUra-induced cytotoxicity was demonstrated. Data suggest that a Fas-dependent component, potentiated by IFN-gamma, exists in FUra/LV-induced cytotoxicity but requires FUra/LV-induced DNA damage for IFN-gamma-induced potentiation to occur.

Inhibition of apoptosis after thymineless stress is conferred by oncogenic K-Ras in colon carcinoma cells.

Ras functions as a molecular switch for several downstream targets and may promote either apoptosis or survival dependent upon the cell system and stimulus. The functional significance of a transfected K-Ras oncogene in influencing apoptosis induced by thymineless stress was examined in a thymidylate synthase (TS)-deficient (TS-) colon carcinoma cell line derived from GC3/c1 after thymidine deprivation. Oncogenic K-Ras conferred survival in TS- K-Ras clones compared with TS- (untransfected) and TS- pCIneo (vector control). Previously, we had demonstrated that thymineless death involved signaling via Fas/FasL interactions. However, in the presence of oncogenic K-Ras, survival did not involve down-regulation of Fas or FasL expression but did involve members of the Bcl-2 family. Bcl-2 and Bax expression remained relatively constant during thymineless stress in all cell lines. Apoptosis in the presence of wild-type Ras correlated with up-regulated expression of Bak that did not occur in TS- K-Ras clones, whereas survival in these clones correlated with elevated expression of Bcl-xL. Thus, the Bak:Bcl-xL ratio was high in TS- and TS- pCIneo cells undergoing apoptosis, whereas the Bcl-xL:Bak ratio was high in TS- K-Ras clones exhibiting a survival response.

Bax is an important determinant of chemosensitivity in pediatric tumor cell lines independent of Bcl-2 expression and p53 status.

Susceptibility of a tumor cell to undergo chemotherapy-induced apoptosis appears to be dependent upon the balance of proapoptotic and survival factors that are expressed within any given cell. We have chosen to evaluate how expression of several of these proteins influences chemosensitivity of a panel of 10 pediatric tumor cell lines chosen from three tumor histiotypes: neuroblastoma, rhabdomyosarcoma, and pediatric glial tumors. The proteins evaluated were p53 and six members of the Bax/Bcl-2 family: three proapoptotic proteins (Bax, Bak, and Bcl-xS) and three survival factors (Bcl-2, Bcl-xL, and Mcl-1). We investigated whether there was any relationship between endogenous expression of these proteins and chemosensitivity (or resistance) to three chemotherapeutic agents that directly damage DNA (doxorubicin, actinomycin D, and topotecan) and a mitotic spindle poison (vincristine). Even though exogenous overexpression of wild-type p53 has been associated with a chemosensitive phenotype in several model systems we demonstrated no such relationship in these studies. In addition, expression levels of Bcl-2, Bcl-xL, Bcl-xS, Bak, or Mcl-1 did not correlate with sensitivity or resistance to the four drugs. However, there was a statistically significant correlation between endogenous levels of Bax protein and sensitivity to both doxorubicin and actinomycin D. We conclude that even though many proteins such as p53 and Bcl-2 have been shown to influence drug response when exogenously overexpressed in model systems, in unmodified cell lines endogenous protein levels may not generate the same results. We have demonstrated that endogenous Bax expression was the only protein found to be associated with chemosensitivity across the three different tumor histiotypes and propose that analysis of Bax may be a more useful prognostic indicator for tumor response to therapy than either p53 or Bcl-2.

Clinical pharmacy anticoagulation services in a group model health maintenance organization.

The clinical pharmacy anticoagulation service (CPAS) at a large group model health maintenance organization is described. The service has expanded dramatically from a local service providing anticoagulation monitoring for the patients of a single physician to a regional service staffed by seven full-time employees who monitor over 3,000 patients. The structure and operations of the CPAS are described as well as the processes used to manage anticoagulation therapy complications. A program for treating patients with deep vein thrombosis in the outpatient setting using enoxaparin is also described.

Genetics of psoriasis: paternal inheritance and a locus on chromosome 6p.

The pathogenesis of psoriasis is not fully understood, but population and twin studies suggest a large heritable component to the etiology. Several large population studies have also suggested a parental sex effect. Since 1994, three main genetic loci (on chromosomes 17q, 4q, and 6p) have been reported in genome scans. With a view to elucidating the genetic basis of psoriasis, we have carried out linkage analysis in a large number of families with well-characterized psoriasis. From a cohort of 1250 probands with psoriasis, 395 individuals (301 affected, 94 unaffected) in 103 families were recruited. Each subject was carefully examined by an experienced dermatologist and stringent diagnostic criteria applied. Genotypes were generated at 11 polymorphic loci on chromosomes 17q, 4q, and 6p and the results were analyzed parametrically and nonparametrically. In the population from which the probands were drawn, there was evidence of a parental sex effect, more probands having an affected father than an affected mother. Genetic anticipation was also apparent and most marked if the disease was inherited from the father. The loci on chromosomes 17 and 4 were not replicated but there was strong evidence for linkage to chromosome 6p (maximum two point LOD score 4.63 at D6S291). The evidence for linkage in sibling pair analysis was greatest when the allele was of paternal origin and was most significant in those families without psoriatic arthritis. These studies confirm the presence of a susceptibility gene on chromosome 6p. The available evidence suggests that a different genetic susceptibility may underlie psoriasis and psoriatic arthritis.